US6703395B2 - Cyclin dependent kinase inhibitor - Google Patents

Cyclin dependent kinase inhibitor Download PDF

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US6703395B2
US6703395B2 US09/816,001 US81600101A US6703395B2 US 6703395 B2 US6703395 B2 US 6703395B2 US 81600101 A US81600101 A US 81600101A US 6703395 B2 US6703395 B2 US 6703395B2
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isopropylpurine
benzylamino
cells
cell
amino
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Libor Havlicek
Miroslav Strnad
Marian Hajduch
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Cyclacel Ltd
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Institute of Experimental Botany Academy CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine

Definitions

  • cdc2 (also known as cdk1) is a catalytic subunit of a family of cyclin dependent kinases that are involved in cell cycle regulation.
  • cyclin dependent kinases comprise at least two subunits, namely a catalytic (of which cdc2 is the prototype) and a regulatory (cyclin).
  • the cdk's are regulated by transitory association with a member of the cyclin family: cyclin A (cdc2, cdk2), cyclin B1-B3 (cdc2), cyclin C (cdk8), cycline D1-D3 (cdk2-cdk4-cdk5-ckd6), cyclin E (cdk2), cyclin H (cdk7).
  • cdk activity is regulated by post-translatory modification, by transitory associations with other proteins, and by modifications of their intracellular localization.
  • the cdk regulators comprise activators (cyclins, cdk7/cyclin H, cdc25 phosphateses), the p9 CKS and p15 cdk-BP sub-units, and the inhibiting proteins (p16 INK4A , p15 INK4B , p21 CiP1 , p18, p27 KiP1 ).
  • the invention relates, in part, to the therapeutic uses of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or other one and pharmaceutically acceptable salts thereof.
  • the present invention pertains to a method of treating a patient suffering from leukemia comprising administering a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or a pharmaceutically acceptable salt thereof.
  • a further aspect of the present invention relates to a method of treating a patient suffering from cancer comprising administering a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or a pharmaceutically acceptable salt thereof.
  • the present invention relates to a method of treating a cancerous or leukemic proliferative disease comprising inhibiting the cdk4 and/or cdk7 enzymes by the administration of a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine (roscovotine) or pharmaceutically acceptable salt thereof.
  • the present invention relates to a method of inducing cell death in proliferative cells comprising administering a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine (roscovotine) or pharmaceutically acceptable salt thereof.
  • FIG. 1 is a representation of in vivo activities of synthetic CDKI's analyzed on models of P338D8 leukemia (FIGS. 1A-1D) and b16 melanoma (FIGS. 1 E- 1 H).
  • FIG. 2 shows cdk4 (FIGS. 2A and 2C) and cdk7 (FIGS. 2B and 2D) activities in vitro and in vivo, when treated with Ip (isopentenyladenine), OC (olomoucine), BOH (bohemine), and ROSC (roscovitine).
  • Ip isopentenyladenine
  • OC olomoucine
  • BOH bihemine
  • ROSC roscovitine
  • FIG. 3 shows that CEM cells induce apoptosis following expression to effective synthetic cdkIs.
  • Leukemia cells were cultured with OC derivatives (50 ⁇ m) for 8 hours and examined by electron microscopy.
  • FIG. 4 shows flow cytometric analysis of the DNA content (FL2) versus the cell size (FS) in CEM cells treated/untreated with various cytokinine derivatives (50 ⁇ m). The data suggest G1/S block followed by extensive apoptosis in the cases of highly effective cdk inhibitors.
  • the invention relates to the therapeutic uses of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or other one and pharmaceutically acceptable salts thereof. It has been observed that these compounds are of particular benefit in the treatment of proliferative diseases such as cancers and leukemia's. As described below these have been observed to possess a mechanism of action not previously reported that provides them with particular benefits, for example, they have been shown to inhibit cell proliferation in a manner that does not induce changes in gene transcription i.e. it functions at a level in the cell cycle regulatory control system that does not involve the regulation of gene expression.
  • the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) is a potent in vivo inhibitor of cdk2, cdk4 and cdk7, properties that provide significant advantages in that this compound is capable of inhibiting cell proliferation in proliferating tissue and not healthy tissue and furthermore is capable of inducing apoptosis (programmed cell death) in proliferative cells.
  • the proliferative cells may be cancer or leukemic cells.
  • the cancer or leukemia is p53 independent.
  • the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) is particulary advantageous for use in the treatment of unwanted cell proliferation because, in contrast to 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine (roscovotine), it does not cause tissue necrosis around the site of injection.
  • a compound of the present invention can be present as a salt, in particular a pharmaceutically acceptable salt.
  • a salt in particular a pharmaceutically acceptable salt.
  • strong inorganic acids such as mineral acids, e.g., sulfuric acid, phosphoric acid or hydrohalic acid
  • organic carboxylic acids such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, e.g., by a halogen, such as acetic acid
  • saturated or unsaturated dicarboxylic acids for example oxalic, malonic, succinic, maleic, fumaric, phthalic or terephthalic acid
  • hydroxycarboxylic acids for example ascorbic, glycolic, lactic, malic, tartaric or citric acid
  • amino acids for example aspartic or glutamic acid
  • benzoic acid or with organic sulfonic acids, such as (C 1 -C 4 )-alkyl- or aryl-sulf
  • the present invention also relates to pharmaceutical compositions comprising 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or a pharmaceutically acceptable salt thereof together with at least one a pharmaceutically acceptable excipient.
  • compositions of the present invention may be adapted for oral, rectal, vaginal, paraenteral, intra-muscular, intra-peritoneal, sub-cutaneous intravenous, nasal or buccal routes of administration.
  • compositions advantageously contain from 1 to 100 mg, and preferably from 10 to 40 mg, of active ingredient per dose.
  • compositions which can be injected intravenously, sub-cutaneously or intra-muscularly, and which are prepared from sterile or sterilizable solutions. They can also be in the form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels and sprays.
  • Injectable forms may contain between 1 and 50 mg, preferably between 10 and 30 mg, of active ingredient per dose.
  • compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dosage, or a multiple or sub-unit of a unit dose.
  • a patient will be administered one or more doses of 10 to 50 mg/day for the treatment of tumors.
  • FTIR spectrum (cm ⁇ 1 ): 1713, 1626, 1572, 1537, 1497, 1471, 1456, 1425, 1398, 1355, 1314, 1292, 1255, 1228, 1202.
  • MTT Thiazolyl blue
  • Human T-lymphoblastic leukemia cell line CEM promyelocytic leukemia HL-60, human breast carcinoma cell line MCF-7, cervical carcinoma cells HELA, mouse fibroblasts NIH3T3, mouse immortalized bone marrow macrophages B2.4 and B10A.4, P388D1 and L1210 leukemias, B16 and B16F10 melanoma were used for routine screening of compounds.
  • the cells were maintained in Nunc/Corning 80 cm 2 plastic tissue culture flasks and cultured in cell culture medium (DMEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 10% fetal calf serum and sodium bicarbonate).
  • DMEM cell culture medium
  • the compound roscovotine 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine was prepared as described in WO97/20842.
  • the compound 2-[(3-hydroxypropyl)amino]-6-benzylamino-9-isopropylpurine prepared as described above, is referred to herein as bohemine.
  • the cell suspensions that were prepared and diluted according to the particular cell type and the expected target cell density (2,500-30,000 cells per well based on cell growth characteristics) were added by pipet (80 ⁇ l) into 96 well microtiter plates. Inoculates were allowed a preincubation period of 24 hours at 37° C. and 5% CO 2 for stabilization. Four-fold dilutions of the intended test concentration were added at time zero in 20 ⁇ l aliquots to the microtiter plate wells. Usually, test compound were evaluated at six 4-fold dilutions. In routine testing, the highest well concentration were 266.7 ⁇ M, but it can be modified dependent on the agent. All drug concentrations were examined in duplicate.
  • Incubations of cells with test compounds lasted for 72 hours at 37° C., in 5% CO 2 atmosphere and 100% humidity.
  • the cells were assayed by using the MTT.
  • Ten microliters of the MTT stock solution were pipeted into each well and incubated further for 1-4 hours.
  • the optical density (OD) was measured at 540 nm with the Labsystem iEMS Reader MF (UK).
  • the TCS 50 value the drug concentration lethal to 50% of the tumor cells, was calculated from the obtained dose response curves.
  • in vivo anti-cancer activity assays water insoluble compounds: 50% DMSO, 10 mM HCl in saline.
  • Mouse melanoma cells B16 were grown in DMEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 10% fetal calf serum and sodium bicarbonate up to 80% confluency. Cells were trypsinized, washed in PBS and suspended in PBS with 0.5% bovine serum albumin. 0.5 miilion cells from this suspension was applied subcutaneously to C57BL-10 mice. One day later, animals were treated with vehicle or indicated compounds: isopentenyladenine (IP), olomoucine (OC), bohemine (BOH), roscovitine (ROSC). The compounds (1 mg) were applied subcutaneously 4 ⁇ daily, for 7 days. Medium survival time (MST) was evaluated in all experimental groups as indicated in FIG. 1 .
  • Mouse P388D1 leukemia was grown in DEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 10% fetal calf serum and sodium bicarbonate up to 80% confluency. Cells were scraped, washed in PBS and suspended in PBS with 0.5% bovine serum albumin. 0.5 million cells from this suspension was applied intraperitoneally to DBA-2 mice. One day later, animals were treated with vehicle or indicated compounds: isopentenyladenine (IP), olomoucine (OC), bohemine (BOH), roscovitine (ROSC). The compounds (1 mg) were applied subcutaneously 4 ⁇ daily, for 7 days. Survival (%) was evaluated in all experimental groups as indicated in FIG. 1 .
  • IP isopentenyladenine
  • OC olomoucine
  • BOH bohemine
  • ROSC roscovitine
  • Cyclin dependent kinases require association with cyclin and phosphorylation on Thr 160/161 for they activity. Cdk 7 was previously identified as an enzyme necessary for this activating phosphorylation.
  • Initial studies on olomoucine derived synthetic CDKIs showed that these compounds inhibit cell cycle progression in late GI phase. Since the activation of cdk4/cdk6 kinases is required for cancer cells to pass the G1 restriction point, we have examined the ability of our compounds to inhibit cdk4 kinase under both in vitro and in vivo conditions.
  • cdk4 was not inhibited with olomoucine derivatives in vitro (IC50>250 ⁇ M) (see Table 2 of WO97/20842), while the activity of cdk4 immunoprecipitated from the cells treated with the same substances was rapidly decreased.
  • cytokinine derived CDKIs down regulate the activity of cdk7 and thus protect cdk4 from activating phosphorylation on Thr161.
  • Glutathione S-transferase-carboxy terminal retinoblastoma protein (GST-Rb) containing multiple phosphorylation sites for cdk2 and cdk4 (kind gift of Dr. Jiri Bartek) was expressed in E. coli and affinity purified.
  • cdc2/cyclin B1 complex was used as a substrate. Its autocatalytic activity was inhibited by covalent binding of ATP analog 5-p-fluorosulfonylbenzoyladenosine (FSBA) to ATP binding site of the kinase.
  • FSBA 5-p-fluorosulfonylbenzoyladenosine
  • baculovirus lysate containing co-expressed cdc2/cyclin B1 were diluted with 1800 ⁇ l of 50 NaCl, 10 mM MgCl 2 , 1 mg/ml ovalbumin, 10% DMSO, 50 mM potassium-HEPES buffer with protease inhibitors, 200 ⁇ l of DMSO containing 20 mM FSBA were added, and the mixture was incubated at room temperature for 30 minutes.
  • the mixture quenched by micro-dialysis against EB buffer (15 mM of MgCl 2 , 20 mM of potassium EGTA, 10 mM of dithiothreitol, 80 mmol of glycerol-2-phosphate, pH 7.3, 1 mM of sodium vanadate, 1 mM of NaF, 1 mM of phenylphosphate, 10 ⁇ g/ml of leupeptin, 10 ⁇ g/ml of aprotinin, 10 ⁇ g/ml of soybean trypsin inhibitor, 100 ⁇ mol of benzamide) overnight at room 4° C.
  • the solution of inactive cdc2/cyclin B1 was concentrated to final volume of 200 ⁇ l by ultrafiltration and stored at ⁇ 80° C. until used.
  • Cdk4 protein was immunoprecipitated from log phase growing cells using the anti-cdk4 monoclonal antibody and Protein-G Sepharose as described elsewhere with only one modification, e.g. two final washes were done in the kinase buffer (60 mmol of glycerol-2-phosphate, 15 mmol of p-nitrophenyl phosphate, 125 mmol of MOPS, pH 7.2, 5 mmol of EGTA, 15 mmol of EGTA, 15 mmol of MgCl 2 , 1 mmol of dithiothreitol, 1 mmol of sodium vanadate, 1 mM of NaF, 1 mmol of phenyl phosphate, 10 ⁇ g/ml of leupeptin, 10 ⁇ g/ml of aprotinin, 10 ⁇ g/ml of soybean trypsin inhibitor, 100 ⁇ mol of benzamide) instead of RIPA buffer.
  • the kinase buffer 60 m
  • immunoprecipitated cdk4 protein equivalent to 10 6 cells in kinase buffer was mixtured on ice with 1 ⁇ l (10 mg/ml) Rb-GST protein, 15 ⁇ mol/ ⁇ - 32 P/ATP (3000 ⁇ Ci/mmol, 1 mCi/ml), 0.3 ⁇ l of DMSO or DMSO solubilized tested compounds at appropriate concentrations in total volume of 30 ⁇ l.
  • Kinase reaction was performed at 30° C. for 15 minutes and it was interrupted by addition of 7 ⁇ l of 5 ⁇ SDS-PAGE sample buffer. Aliquots of these samples (25 ⁇ l) were electrophoresed in 10% polyacrylamide gels, autoradiographed and quantified by densitometry.
  • Cdk7 protein was immunoprecipitated from log phase growing CEM cells using the anti-cdk7 monoclonal antibody and Protein-G Sepharose as described elsewhere, with only one modification, e.g. two final washes were done in the EB buffer instead of RIPA buffer.
  • immunoprecipitated cdk7 protein equivalent to 10 6 cells was mixtured on ice with 10 ⁇ l of FSBA inactivated cdc2/cyclin B1 complex as a substrate, 3 ⁇ l of 1M MgCl 2 , 15 ⁇ mol/ ⁇ - 32 P/ATP (3000 ⁇ Ci/mmol, 1 mCi/ml), and 1 ⁇ l DMSO and/or DMSO solutions of tested compounds at appropriate concentrations respectively in total volume of 100 ⁇ l of EB buffer. This reaction mixture was incubated at 30° C. and reaction was quenched after 30 minutes by addition of 20 ⁇ l 5 ⁇ SDS-PAGE sample buffer. 25 ⁇ l aliquots of the samples were electrophoresed in 12% polyacrylamide gels, autoradiographed and quantified by densitometry.
  • Phase contrast microscopy and follow-up electron microscopy confirmed that apoptosis had occurred (see FIG. 3 for electron micrograph of olomucine, roscovotine and bohemin treated cells).
  • Flow cytometric analysis of DNA content in CEM cells showed similar results. Extensive apoptosis was noted in roscovotine and bohemin treated cells (FIG. 4 ).

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Abstract

The present invention is based on the method of treating a patient suffering from leukemia comprising administering a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine or a pharmaceutically acceptable salt thereof.

Description

RELATED APPLICATIONS
This application is a continuation application of Ser. No. 09/118,247 filed on Jul. 17, 1998, now abandoned, which in turn is a continuation-in-part application of Ser. No. 09/034,581 filed on Mar. 4, 1998, now U.S. Pat. No. 6,221,873. The contents of all of the aforementioned application(s) are hereby incorporated by reference.
BACKGROUND
The prior art has described several compounds that are capable of regulating the cell cycle by virtue of inhibiting cyclin dependent kinases. These compounds include butyrolactone, flavopiridol and 2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine (olomoucin). Olomucin and related compounds have been shown to be inhibitors of cdc2. cdc2 (also known as cdk1) is a catalytic subunit of a family of cyclin dependent kinases that are involved in cell cycle regulation.
These cyclin dependent kinases comprise at least two subunits, namely a catalytic (of which cdc2 is the prototype) and a regulatory (cyclin). The cdk's are regulated by transitory association with a member of the cyclin family: cyclin A (cdc2, cdk2), cyclin B1-B3 (cdc2), cyclin C (cdk8), cycline D1-D3 (cdk2-cdk4-cdk5-ckd6), cyclin E (cdk2), cyclin H (cdk7).
Each of these complexes is involved in a phase of the cellular cycle. cdk activity is regulated by post-translatory modification, by transitory associations with other proteins, and by modifications of their intracellular localization. The cdk regulators comprise activators (cyclins, cdk7/cyclin H, cdc25 phosphateses), the p9CKS and p15cdk-BP sub-units, and the inhibiting proteins (p16INK4A, p15INK4B, p21CiP1, p18, p27KiP1).
There is now considerable support in the literature for the hypothesis that cdk's and their regulatory proteins play a significant role in the development of human tumors. Thus, in numerous tumors a temporal abnormal expression of cyclin-dependent kinases, and a major deregulation of protein inhibitors (mutations, deletions) has been observed.
SUMMARY
The invention relates, in part, to the therapeutic uses of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or other one and pharmaceutically acceptable salts thereof.
The present invention pertains to a method of treating a patient suffering from leukemia comprising administering a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or a pharmaceutically acceptable salt thereof.
A further aspect of the present invention relates to a method of treating a patient suffering from cancer comprising administering a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or a pharmaceutically acceptable salt thereof.
In a further embodiment the present invention relates to a method of treating a cancerous or leukemic proliferative disease comprising inhibiting the cdk4 and/or cdk7 enzymes by the administration of a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine (roscovotine) or pharmaceutically acceptable salt thereof.
In an additional embodiment the present invention relates to a method of inducing cell death in proliferative cells comprising administering a therapeutically effective amount of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine (roscovotine) or pharmaceutically acceptable salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a representation of in vivo activities of synthetic CDKI's analyzed on models of P338D8 leukemia (FIGS. 1A-1D) and b16 melanoma (FIGS. 1E-1H).
FIG. 2 shows cdk4 (FIGS. 2A and 2C) and cdk7 (FIGS. 2B and 2D) activities in vitro and in vivo, when treated with Ip (isopentenyladenine), OC (olomoucine), BOH (bohemine), and ROSC (roscovitine).
FIG. 3 shows that CEM cells induce apoptosis following expression to effective synthetic cdkIs. Leukemia cells were cultured with OC derivatives (50 μm) for 8 hours and examined by electron microscopy.
FIG. 4 shows flow cytometric analysis of the DNA content (FL2) versus the cell size (FS) in CEM cells treated/untreated with various cytokinine derivatives (50 μm). The data suggest G1/S block followed by extensive apoptosis in the cases of highly effective cdk inhibitors.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to the therapeutic uses of the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or other one and pharmaceutically acceptable salts thereof. It has been observed that these compounds are of particular benefit in the treatment of proliferative diseases such as cancers and leukemia's. As described below these have been observed to possess a mechanism of action not previously reported that provides them with particular benefits, for example, they have been shown to inhibit cell proliferation in a manner that does not induce changes in gene transcription i.e. it functions at a level in the cell cycle regulatory control system that does not involve the regulation of gene expression.
The compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) is a potent in vivo inhibitor of cdk2, cdk4 and cdk7, properties that provide significant advantages in that this compound is capable of inhibiting cell proliferation in proliferating tissue and not healthy tissue and furthermore is capable of inducing apoptosis (programmed cell death) in proliferative cells. In certain embodiments, the proliferative cells may be cancer or leukemic cells. In a preferred embodiments, the cancer or leukemia is p53 independent.
The compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) is particulary advantageous for use in the treatment of unwanted cell proliferation because, in contrast to 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine (roscovotine), it does not cause tissue necrosis around the site of injection.
Administration of Compounds
A compound of the present invention can be present as a salt, in particular a pharmaceutically acceptable salt. These are formed, for example, with strong inorganic acids, such as mineral acids, e.g., sulfuric acid, phosphoric acid or hydrohalic acid; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, e.g., by a halogen, such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or terephthalic acid; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with amino acids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted, for example, by a halogen such as methane- or p-toluene sulfonic acid.
The present invention also relates to pharmaceutical compositions comprising 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine (bohemine) or a pharmaceutically acceptable salt thereof together with at least one a pharmaceutically acceptable excipient.
The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, paraenteral, intra-muscular, intra-peritoneal, sub-cutaneous intravenous, nasal or buccal routes of administration.
For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops and capsules. These compositions advantageously contain from 1 to 100 mg, and preferably from 10 to 40 mg, of active ingredient per dose.
Other forms of administration comprise solutions which can be injected intravenously, sub-cutaneously or intra-muscularly, and which are prepared from sterile or sterilizable solutions. They can also be in the form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels and sprays.
Injectable forms may contain between 1 and 50 mg, preferably between 10 and 30 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dosage, or a multiple or sub-unit of a unit dose.
One of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. In an exemplary embodiment, a patient will be administered one or more doses of 10 to 50 mg/day for the treatment of tumors.
The following invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. The contents of WO97/20842 (PCT/FR96/01905) are also incorporated herein by reference.
EXAMPLES Example 1 Preparation of 2-([(3-hydroxypropyl)amino]-6-benzylamino-9-isopropylpurine (Bohemine) Materials and Methods
Melting points were determined on a Kofier block and are uncorrected. Evaporations were carried out with a rotary evaporator under water-pump vacuum or rotary oil pump in the case of dimethyl sulfoxide and amines. The ′H NMR spectra (δ, ppm; J, Hz) were measured on Varian VXR-400(400 MHz) or on Varian Unity 200(200 MHz). All spectra were obtained at 25° C. using tetramethylsilane as internal standard. Electron impact mass spectra (m/z, rel. %) were measured on a VG 7070E spectrometer (70 e/V 200° C., direct inlet) or on a Finnigan MAT 90 spectrometer (70 eV, 250° C., direct inlet) or on a Jeol JMS-D100(80 eV, 200° C., direct inlet). High resolution measurements were carried out by the peak-matching method using Ultramarck 1600F(PCR Inc., FL, USA) as a standard. The instrument was tuned to a resolution of 8,000 (10% valley definition). Infrared spectra were recorded on a FTIR Nicolet 205 instrument as KBr disks. Merck silica gel Kieselgel 60 (230-400 mesh) was used for column chromatography. TLC was carried out on Merck DC Alufolien Kieselgel 60 F254 plates.
6-Benzylamino-2-chloro-9-isopropylpurine (2)
A mixture of 6-benzylamino-2-chloropurine (Hocart C. H., Letham D. S., Parker C. W.: Phytochemistry 30,2477 (1991)) 1.32 g, 5.08 mmol), potassium carbonate (4.3 g, 31 mmol) and isopropylbromide (5.5 ml, 58 mmol) in 35 ml of dry dimethyl sulfoxide was vigorously shaken overnight. The reaction mixture was evaporated in vacuum and then partitioned between water and ethylacetate. The organic layer was dried (sodium sulfate) and evaporated. Crystallization from methanol afforded 1.305 g (85%) of product 2, m.p. 181-182° C.
For C15H16N5Cl(301.78) calculated: 59.70% C, 5.34% H, 23.21% N, 11.75% Cl; found: 59.52% C, 5.36% H, 23.01% N.
FTIR spectrum (cm−1): 1713, 1626, 1572, 1537, 1497, 1471, 1456, 1425, 1398, 1355, 1314, 1292, 1255, 1228, 1202.
′H NMR (400 MHz, (CH3)2SO): 1.48D (6H, J=6.8, (CH3)2CH); 4.57m (1H, CH(CH3)2); 4.66bd (2H, J=6.1, CH2); 7.19ft, (1 H, J=7.2, J=1.7, H-p); 7.27dd (2H, J=7.2, H-m); 7.34dd (2H, J=7.2, J=1.7, H-o); 7.69s (1 H, H-C8).
2-[(3-hydroxypropyl)amino]-6-benzylamino-9-isopropylpurine (3)
2-Chloroderivative (2) (0.5 mmol) and 3 ml of 3-aminopropanol were heated for 3 hours to 160° C. (sealed ampoule). Excess of the amine was evaporated at a temperature below 70° C. and the residue was purified by column chromatography (stepwise 0; 1; 2% MEOH in CHCl3) and crystallized from diethyl ether afforded the product in 82% yield; m.p. 98-101° C.
For C18H24N60(340.43) calc. 63.51% C, 7.11% H, 24.69% N; found 63.43% C, 7.05% H, 24.60% N,
Mass spectrum (Finnigan MAT 90): 341 (21), 340.21 10 (M+−, C18H24N60 calc. 340.2012, 100%), 339 (4), 310 (9), 309 (33), 297 (11), 296 (28), 295 (38), 282 (9), 253 (6), 251 (4), 239 (4), 191 (6), 134 (6), 106 (8), 92 (4), 91 (44), 43 (9), 41(7).
′H NMR (200 MHz, CDC13): 1.53d (6H, J=7, (CH3)2CH); 1.68-1.81m (2H, CH2CH2CH2); 3.55-3.71M (4H, CH2N+CH20); 4.62hept (1H, J=7, CH(CH3)2); 4.76bd (2H, J=4.5, CH2Ph); 4.96bt (1H, NHC2); 5.10bs (OH or NH); 6.00bs (NH, or OH), 7.22-7.38m (5H, Ph), 7.47s (1 H, H±C8).
Example 2 An Alternative Synthesis or 6-benzylamino-2-chloro-9-isopropylpurine (2)
2.6-dichloro-9-isopropylpurine
A mixture of 2,6-dichloropurine 1 g (5.3 mmol), powdered potassium carbonate 2 g (14 mmol) were vigorously stirred in 35 ml DMF. Isopropyliodide 3 ml (30 mmol) was added in five portions within 10 hours. Monitoring of the reaction by TLC (EtOAc/heptaine; 1/1) showed that the reaction was nearly complete.
The principle product was isolated by column chromatography stepwise 20, 30, 40% EtOAc in heptaine; crystalization EtOAc/pentane; yield 0.54 g (44%); m.p. 148-150′C.
For C8H8N4C12 Calc: 41.58; H, 3.49; N, 24.25; Cl 30.68. Found: C, 41.29; H, 3.71, N, 24.01
′H NMR (400 MHz, CD30D): 1.67(6H, d, J=6.8, (CH3)2CH), 4.93(1H, mt, CH(CH3)2), 8.67(1 H, s, H_C8).
13C NMR (100 MHz, CD30D): 22.74Qqd (J=128.0, J=4.6, J=4.6), 50.79
Dsep (J=143.4, J=4.4), 132.41d (J=11.6)147.67 Dd (J=214.0, J=4.4), 152.24 s, 153.91 s, 154.75 br mt.
6-benzylamino-2-chloro-9-isopropylpurine (2)
2,6-dichloro-9-isopropylpurine (1 mmol) and benzylamine (3.5 mmol) were heated with stirring in 3 ml of n-butanol (3 hour, 110° C.). The reaction mixture (after evaporation of n-butanol) was worked up as above for the compound 2, yield 85-90%.
Example 3 In vitro Cytotoxic Activity of (Bohemine) 2-[(3-hydroxypropyl)amino]-6-benzylamino-9-isopropylpurine Introduction
One of the parameters used as the basis for colorimetric assays is the metabolic activity of viable cells. For example, a microtiter assay which uses the tetrazolium salt 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue (hereinafter MTT) is now widely used to quantitate cell proliferation and cytotoxicity, for instance, in drug screening programs and in chemosensitivity testing. Because tetrazolium salts are cleaved only by metabolically active cells, these assays detect viable cells exclusively. In the case of the MTT-assay, yellow soluble tetrazolium salt is reduced to colored water-insoluble formazan salt. After it is solubilized, the formazan formed can easily and rapidly be quantified in a conventional ELISA plate reader at 570 nm (maximum absorbance). The quantity of reduced formazan corresponds to number of vital cells in the culture.
Materials
The formulation material used in the
in vitro cyctotoxicity/antiproliferative assays: 10% dimethylsulfoxide (DMSO), 80 mM HCl in physiological saline (0.9%) w/v sodium chloride)
Human T-lymphoblastic leukemia cell line CEM, promyelocytic leukemia HL-60, human breast carcinoma cell line MCF-7, cervical carcinoma cells HELA, mouse fibroblasts NIH3T3, mouse immortalized bone marrow macrophages B2.4 and B10A.4, P388D1 and L1210 leukemias, B16 and B16F10 melanoma were used for routine screening of compounds. The cells were maintained in Nunc/Corning 80 cm2 plastic tissue culture flasks and cultured in cell culture medium (DMEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal calf serum and sodium bicarbonate). The compound roscovotine 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamino-9-isopropylpurine was prepared as described in WO97/20842. The compound 2-[(3-hydroxypropyl)amino]-6-benzylamino-9-isopropylpurine prepared as described above, is referred to herein as bohemine.
Methods
The cell suspensions that were prepared and diluted according to the particular cell type and the expected target cell density (2,500-30,000 cells per well based on cell growth characteristics) were added by pipet (80 μl) into 96 well microtiter plates. Inoculates were allowed a preincubation period of 24 hours at 37° C. and 5% CO2 for stabilization. Four-fold dilutions of the intended test concentration were added at time zero in 20 μl aliquots to the microtiter plate wells. Usually, test compound were evaluated at six 4-fold dilutions. In routine testing, the highest well concentration were 266.7 μM, but it can be modified dependent on the agent. All drug concentrations were examined in duplicate. Incubations of cells with test compounds lasted for 72 hours at 37° C., in 5% CO2 atmosphere and 100% humidity. At the end of incubation period, the cells were assayed by using the MTT. Ten microliters of the MTT stock solution were pipeted into each well and incubated further for 1-4 hours. After this incubation period, formazan was solubilized by addition of 100 μl/well of 10% SDS in water (pH=5.5) followed by further incubation at 37° C. overnight. The optical density (OD) was measured at 540 nm with the Labsystem iEMS Reader MF (UK). The tumor cell survival (TCS) was calculated using the following equation: TCS=(ODdrug exposed well/mean ODcontrol wells)×100%. The TCS50 value, the drug concentration lethal to 50% of the tumor cells, was calculated from the obtained dose response curves.
Results
In vitro Cytotoxic Activity of Bohemine
To evaluate anti-cancer activity of IP, OC, BOH and ROSCa, toxicities of these compounds were tested on panel of cell lines of different histogenetic and species origin (Table 1).
TABLE 1
IC50 (μM)
CEM HL-60 B16 B16F10 MCF7 HELA B10# 4 B2#4 U937 L1210 P388D1 NIH3T3 Lymphocytes
IP 168.9 157.41 266.7 266.7 266.7 266.7 266.7 266.7 266.7 266.7 266.7 266.7 266.7
OC 20.68 28.9 50.4 20.6 30.53 100.4 20.26 8.1 63.8 23.5 14.5 266.7 266.7
BOH 6.16 16.7 22.6 4.3 17.0 11.1 8.3 6.4 20.1 12.8 6.3 266.7 266.7
ROSC 4.3 11.7 15.8 4.0 5.27 7.55 15.9 6.9 19.4 8.7 5.8 266.7 266.7
Equal activities were found in all tumor cell lines tested, however, the non-malignant cell, e.g. NIH3T3 fibroblasts and normal human lymphocytes, were resistant to synthetic CDKI induced toxicity. As demonstrated in Table 1, IP did not show significant toxicity, OC was moderately effective, while BOH and ROSC killed tumor cells in concentrations close to 10-20 μM.
Example 4 In vivo Antitumor Activity of Bohemine
To analyze the effects of cytokinine derivatives on growth of transplanted tumors, the models of intraperitoneally transplanted P388D1 leukemia (4×1 mg/day i.p.) and subcutaneously innoculated B16 melanoma were applied.
The formulation material used in the
in vivo anti-cancer activity assays: water insoluble compounds: 50% DMSO, 10 mM HCl in saline.
Mouse Melanoma B16
Mouse melanoma cells B16 were grown in DMEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal calf serum and sodium bicarbonate up to 80% confluency. Cells were trypsinized, washed in PBS and suspended in PBS with 0.5% bovine serum albumin. 0.5 miilion cells from this suspension was applied subcutaneously to C57BL-10 mice. One day later, animals were treated with vehicle or indicated compounds: isopentenyladenine (IP), olomoucine (OC), bohemine (BOH), roscovitine (ROSC). The compounds (1 mg) were applied subcutaneously 4× daily, for 7 days. Medium survival time (MST) was evaluated in all experimental groups as indicated in FIG. 1.
Murine Leukemia P388D1
Mouse P388D1 leukemia was grown in DEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal calf serum and sodium bicarbonate up to 80% confluency. Cells were scraped, washed in PBS and suspended in PBS with 0.5% bovine serum albumin. 0.5 million cells from this suspension was applied intraperitoneally to DBA-2 mice. One day later, animals were treated with vehicle or indicated compounds: isopentenyladenine (IP), olomoucine (OC), bohemine (BOH), roscovitine (ROSC). The compounds (1 mg) were applied subcutaneously 4× daily, for 7 days. Survival (%) was evaluated in all experimental groups as indicated in FIG. 1.
Results
In contrast to in vitro assays, only olomoucine and bohemine showed significant in vivo activity in both mouse P388D1 leukemia and B16 melanoma models (FIG. 1). Isopentenyladenine showed no activity and toxicity in vivo, while roscovitine was inactive and, moreover, its application resulted in induction of deep skin/peritoneal necrotic lesions after subcutaneous/intraperitoneal application. These results indicate that enzyme inhibitory activity and in vitro cytotoxicity of CDKIs does not automatically predict anti-cancer activity.
Example 5 The Effect of Olomoucine Derived Synthetic CDKIs on cdk4 and cdk7 Activities Under in vitro and in vivo Conditions Introduction
Cyclin dependent kinases require association with cyclin and phosphorylation on Thr 160/161 for they activity. Cdk 7 was previously identified as an enzyme necessary for this activating phosphorylation. Initial studies on olomoucine derived synthetic CDKIs showed that these compounds inhibit cell cycle progression in late GI phase. Since the activation of cdk4/cdk6 kinases is required for cancer cells to pass the G1 restriction point, we have examined the ability of our compounds to inhibit cdk4 kinase under both in vitro and in vivo conditions. In agreement with previous observations, cdk4 was not inhibited with olomoucine derivatives in vitro (IC50>250 μM) (see Table 2 of WO97/20842), while the activity of cdk4 immunoprecipitated from the cells treated with the same substances was rapidly decreased. One of the possible explanations was that cytokinine derived CDKIs down regulate the activity of cdk7 and thus protect cdk4 from activating phosphorylation on Thr161. It is described here for the first time that the compound 2-([(3-hydroxypropyl)amino]-6-benzylamino)-9-isopropylpurine is a poten* inhibitor of cdk7 under both in vitro and in vivo conditions where cdk4 inhibition is also observed. This family of compounds can, thus, be used generally for the treatment of tumors with increased activity of cdk7 and/or cdk4.
Protein Reagents
Baculovirus encoded human cdc2 and cyclin B1, (kind gift of Dr. David Lane) were expressed in Sf9 cells as described elsewhere. Glutathione S-transferase-carboxy terminal retinoblastoma protein (GST-Rb) containing multiple phosphorylation sites for cdk2 and cdk4 (kind gift of Dr. Jiri Bartek) was expressed in E. coli and affinity purified.
For cdk7 activity studies, cdc2/cyclin B1 complex was used as a substrate. Its autocatalytic activity was inhibited by covalent binding of ATP analog 5-p-fluorosulfonylbenzoyladenosine (FSBA) to ATP binding site of the kinase. Briefly, 200 μl of baculovirus lysate containing co-expressed cdc2/cyclin B1 were diluted with 1800 μl of 50 NaCl, 10 mM MgCl2, 1 mg/ml ovalbumin, 10% DMSO, 50 mM potassium-HEPES buffer with protease inhibitors, 200 μl of DMSO containing 20 mM FSBA were added, and the mixture was incubated at room temperature for 30 minutes. Following the incubation period, the mixture quenched by micro-dialysis against EB buffer (15 mM of MgCl2, 20 mM of potassium EGTA, 10 mM of dithiothreitol, 80 mmol of glycerol-2-phosphate, pH 7.3, 1 mM of sodium vanadate, 1 mM of NaF, 1 mM of phenylphosphate, 10 μg/ml of leupeptin, 10 μg/ml of aprotinin, 10 μg/ml of soybean trypsin inhibitor, 100 μmol of benzamide) overnight at room 4° C. The solution of inactive cdc2/cyclin B1 was concentrated to final volume of 200 μl by ultrafiltration and stored at −80° C. until used.
cdk4 Activity
Cdk4 protein was immunoprecipitated from log phase growing cells using the anti-cdk4 monoclonal antibody and Protein-G Sepharose as described elsewhere with only one modification, e.g. two final washes were done in the kinase buffer (60 mmol of glycerol-2-phosphate, 15 mmol of p-nitrophenyl phosphate, 125 mmol of MOPS, pH 7.2, 5 mmol of EGTA, 15 mmol of EGTA, 15 mmol of MgCl2, 1 mmol of dithiothreitol, 1 mmol of sodium vanadate, 1 mM of NaF, 1 mmol of phenyl phosphate, 10 μg/ml of leupeptin, 10 μg/ml of aprotinin, 10 μg/ml of soybean trypsin inhibitor, 100 μmol of benzamide) instead of RIPA buffer. Following the final wash, immunoprecipitated cdk4 protein equivalent to 106 cells in kinase buffer was mixtured on ice with 1 μl (10 mg/ml) Rb-GST protein, 15 μmol/γ-32P/ATP (3000 μCi/mmol, 1 mCi/ml), 0.3 μl of DMSO or DMSO solubilized tested compounds at appropriate concentrations in total volume of 30 μl. Kinase reaction was performed at 30° C. for 15 minutes and it was interrupted by addition of 7 μl of 5×SDS-PAGE sample buffer. Aliquots of these samples (25 μl) were electrophoresed in 10% polyacrylamide gels, autoradiographed and quantified by densitometry.
cdk7 Activity
Cdk7 protein was immunoprecipitated from log phase growing CEM cells using the anti-cdk7 monoclonal antibody and Protein-G Sepharose as described elsewhere, with only one modification, e.g. two final washes were done in the EB buffer instead of RIPA buffer. Following the final wash, immunoprecipitated cdk7 protein equivalent to 106 cells was mixtured on ice with 10 μl of FSBA inactivated cdc2/cyclin B1 complex as a substrate, 3 μl of 1M MgCl2, 15 μmol/γ-32P/ATP (3000 μCi/mmol, 1 mCi/ml), and 1 μl DMSO and/or DMSO solutions of tested compounds at appropriate concentrations respectively in total volume of 100 μl of EB buffer. This reaction mixture was incubated at 30° C. and reaction was quenched after 30 minutes by addition of 20 μl 5× SDS-PAGE sample buffer. 25 μl aliquots of the samples were electrophoresed in 12% polyacrylamide gels, autoradiographed and quantified by densitometry.
Results
The results demonstrate that cytokinine derivatives were found to inhibit cdk4 activity in vivo, while the same compounds were entirely ineffective under in vitro conditions. This observation could be explained by dysregulation of Try15 dephosphorylation process and cdc25 phosphatase activity. The inhibition of cdk7 is responsible for decrease in cdk4 activity in vivo. This is the first demonstration of ability of OC derived synthetic CDKIs to inhibit cdk7 kinase under both in vitro and in vivo conditions.
Example 6 Apoptosis and Cell Cycle Analysis by FACS
Cells (1×106/ml) were cultured in 6-well plates with or without 70 μM concentration of test compound at 37° C. and 5% CO2 for 3-24 hours. Following incubation, cells were pelleted, washed in Hank's buffered salt solution and fixed in 96% ethanol overnight at −20° C. Low molecular weight apoptotic DNA was extracted in citrate buffer. RNA was cleaved using RNAse (50 mg/ml). DNA was stained by ethidium bromide, and cells were analysed in FACS using a 488 nm single beam laser (Coulter).
Results
Phase contrast microscopy and follow-up electron microscopy confirmed that apoptosis had occurred (see FIG. 3 for electron micrograph of olomucine, roscovotine and bohemin treated cells). Flow cytometric analysis of DNA content in CEM cells showed similar results. Extensive apoptosis was noted in roscovotine and bohemin treated cells (FIG. 4).
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (12)

What is claimed is:
1. A method of inducing apoptosis in a cell, comprising intentionally contacting said cell with an effective amount of the compound 2-({(1-ethyl-2-hydroxyethyl)amino}-6-benzylamino)-9-isopropylpurine or a pharmaceutically acceptable salt thereof, such that apoptosis is induced in said cell.
2. The method according to claim 1, wherein the cell is a cancer or leukemia cell.
3. The method according to claim 2, wherein the leukemia or cancer is not dependent on p53.
4. A method of treating a proliferative disease comprising inducing apoptosis in a subject in need thereof, by the administration of a therapeutically effective amount of the compound 2-({(1-ethyl-2-hydroxyethyl)amino}-6-benzylamino)-9-isopropylpurine or a pharmaceutically acceptable salt thereof to said subject, such that said proliferative disease is treated.
5. The method of claim 1, wherein said apoptosis is induced by inhibition of cdk4 or cdk7 enzymes.
6. The method of claim 4, wherein cdk4 or cdk7 enzymes are inhibited in said subject.
7. The method of claim 6, wherein said compound induces cell death in proliferative cells that are not dependent on p53.
8. The method of claim 7, wherein said proliferative cells are cancer or leukemic cells.
9. A method of treating a proliferative disease associated with increased cdk7 or cdk4 activity, comprising administering to a subject in need thereof a therapeutically effective amount of the compound 2-({(1-ethyl-2-hydroxyethyl)amino}-6-benzylamino)-9-isopropylpurine or a pharmaceutically acceptable salt thereof, such that said proliferative disease in said subject is treated.
10. The method of claim 9, wherein said compound 2-({(1-ethyl-2-hydroxyethyl)amino}-6-benzylamino)-9-isopropylpurine or a pharmaceutically acceptable salt thereof is administered in an amount sufficient to induce apoptosis.
11. The method of claim 9 or 10, wherein said proliferative disease is a cancer or leukemia.
12. The method of claim 11, wherein said cancer or leukemia is not dependent on p53.
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